Research on the effect of selective kinase inhibitors on neutrophile activation

Epidermolysis Bullosa Acquisita (EBA) belongs to the pemphigoid diseases, which are a group of autoimmune blistering diseases of the skin and mucous membranes. They often show severe clinical symptoms and a chronic disease progression (Schmidt, E. und Zillikens, D., 2013). So far, the first line treatment is still corticosteroids, which can cause severe side effects. In EBA, the skin damages are caused by an autoimmune reaction which is directed against the collagen 7 (Col7) of the dermal-epidermal junction. The formation of the immunocomplex (IC) between the autoantigen and antibody results in the migration and activation of immune cells. Especially neutrophilic cells directly contribute to split formation by the release of reactive oxygen species (ROS) and proteases (Koga, H. et al., 2019 und Kim, J.H. et al. 2011) ). We therefore hypothesize that a selective inhibition of kinases involved in IC activation could be a promising target strategy for patients with pemphigoid diseases. To get a comprehensive overview on the neutrophilic kinase network, we performed PamGene kinome analysis of IC- activated neutrophils. Several kinases have not been associated with IC-induced signal transduction within neutrophils and will be further studied here. (i) Investigation of the inhibitory effect of several kinase inhibitors on neutrophil activation (ii) Determination of neutrophilic cell toxicity induced by these kinase inhibitors (iii) Analysis of the effect of the kinase inhibitors on certain cell surface markers of activated neutrophils. Neutrophils from healthy blood donors will be isolated and activated with ICs of recombinant human Col7 and anti-Col7 IgG1. In parallel, a total of 11 different kinase inhibitors in four different concentrations is added to the activated neutrophils.To address objective (i) we are using a ROS release assay.  Once the neutrophils are activated, they release ROS, which is measured by luminol chemiluminescence. To analyze objective (ii) and (iii) we will perform FACS analysis (fluorescence activated cell sorting). To check for the cell toxicity induced by kinase inhibitors, we will stain the neutrophils with Annexin V, which binds to the apoptotic cell surface marker phosphatidylserine, and ZombieNIR, which intercalates with DNA in necrotic cells. Additionally, neutrophils will be stained with antibodies against CD18 and CD62L. CD18 is a degranulating marker, thus, it is highly expressed on activated cells. CD62L is a cell adhesion molecule and belongs to the selectin family. When neutrophils are activated, CD62L is shedded which can be detected by FACS.